WO2009034414A1 - Method for fermenting cellulosics - Google Patents
Method for fermenting cellulosics Download PDFInfo
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- WO2009034414A1 WO2009034414A1 PCT/IB2007/004098 IB2007004098W WO2009034414A1 WO 2009034414 A1 WO2009034414 A1 WO 2009034414A1 IB 2007004098 W IB2007004098 W IB 2007004098W WO 2009034414 A1 WO2009034414 A1 WO 2009034414A1
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- host cell
- recombinant host
- glucosidase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
- C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- the present invention relates to the field of metabolic engineering. Particularly, the invention relates to engineering organisms to ferment biomass material for the production of ethanol.
- Plant biomass consists of about 40-55% cellulose, 25-50% hemicellulose and 10-40% lignin, depending whether the source is hardwood, softwood, or grasses (Sun, Y. and Cheng, J., Bioresource Technol. 83, 1-11 (2002)).
- the major polysaccharide present is water-insoluble cellulose that contains the major fraction of fermentable sugars (glucose, cellobiose or cellodextrins).
- Native cellulose consists of amorphous and crystalline regions ( Figure 1).
- endoglucanases (1,4- ⁇ -D-glucan 4-glucanohydrolases; EC 3.2.1.4)
- exoglucanases including cellodextrinases (1,4- ⁇ -D-glucan glucanohydrolases; EC 3.2.1.74) and cellobiohydrolases (1,4- ⁇ -D-glucan cellobiohydrolases; EC 3.2.1.91);
- ⁇ -glucosidases ⁇ -glucoside glucohydrolases; EC 3.2.1.21).
- Endoglucanases cut at random in the cellulose polysaccharide chain of amorphous cellulose, generating oligosaccharides of varying lengths and consequently new chain ends.
- Exoglucanases act in a processive manner on the reducing or non-reducing ends of cellulose polysaccharide chains, liberating either glucose (glucanohydrolases) or cellobiose (cellobiohydrolase) as major products. Exoglucanases can also act on microcrystalline cellulose, presumably peeling cellulose chains from the microcrystalline structure.
- ⁇ -Glucosidases hydrolyze soluble cellodextrins and cellobiose to glucose units ( Figure 1).
- a variety of plant biomass resources are available as lignocellulosics for the production of biofuels, notably bioethanol.
- the major sources are (i) wood residues from paper mills, sawmills and furniture manufacturing, (ii) municipal solid wastes, (iii) agricultural residues and (iv) energy crops.
- Pre-conversion of particularly the cellulosic fraction in these biomass resources using either physical, chemical or enzymatic processes) to fermentable sugars (glucose, cellobiose and cellodextrins) would enable their fermentation to bioethanol, provided the necessary fermentative micro-organism with the ability to utilize these sugars is used.
- Bakers' yeast ( ⁇ accharomyces cerevisiae) remains the preferred micro-organism for the production of ethanol (Hahn-Hagerdal, B., et al, Adv. Biochem. Eng. Biotechnol. 73, 53—84 (2001)). Attributes in favor of this microbe are (i) high productivity at close to theoretical yields (0.51 g ethanol produced / g glucose used), (ii) high osmo- and ethanol tolerance, (iii) natural robustness in industrial processes, and (iv) being generally regarded as safe due to its long association with wine and bread making, and beer brewing.
- the major shortcoming of S. cerevisiae is its inability to utilize complex polysaccharides such as cellulose, or its break-down products, such as cellobiose and cellodextrins.
- Fujita, Y., et al attempted to extend this previous work and engineer yeast to ferment cellulosic material by immobilizing various enzymes on the yeast surface.
- Fujita et al. were unable to achieve fermentation of amorphous cellulose using yeast expressing only recombinant endoglucanase and ⁇ -glucosidase.
- Another limitation of the Fujita et al approach was that cells had to be pre-grown to high cell density on standard carbon sources before the cells were useful for ethanol production using amorphous cellulose.
- Fujita et al. teach high biomass loadings of —15 g/L to accomplish ethanol production.
- recombinant host cells are able to grow and multiply on amorphous cellulose and concomitantly produce ethanol. This improvement brings about drastic cost savings and increased industrial efficiency by not requiring extensive pre-growth of the cells.
- Another aspect of the invention relates to a recombinant host cell comprising a heterologous polynucleotide which encodes an endoglucanase and a heterologous polynucleotide which encodes a ⁇ -glucosidase, wherein the host cell does not express an exoglucanase, and wherein the host cell can grow on amorphous cellulose as the sole carbon source.
- Another aspect of the invention relates to a recombinant host cell comprising a heterologous polynucleotide which encodes an endoglucanase and a heterologous polynucleotide which encodes a ⁇ -glucosidase, wherein the host cell can grow on amorphous cellulose as the sole carbon source, and wherein the host cell does not require pre-growth on a non-amorphous cellulose carbon source.
- a further aspect of the present invention relates to a recombinant host cell comprising a heterologous polynucleotide which encodes an endoglucanase and a heterologous polynucleotide which encodes a ⁇ -glucosidase that can grow on amorphous cellulose as the sole carbon source and concomitantly produce ethanol.
- Yet another aspect of the invention relates to methods for direct fermentation of amorphous cellulosics to ethanol, utilizing a recombinant yeast strain producing both endoglucanase and ⁇ -glucosidase enzymes.
- methods of producing ethanol comprise contacting a composition comprising amorphous cellulose with a recombinant host cell wherein the host cell ferments amorphous cellulose to ethanol and then recovering the ethanol.
- compositions comprising recombinant host cells capable of fermenting cellulosic material.
- Figure 1 depicts a schematic representation of the hydrolysis of amorphous and microcrystalline cellulose by noncomplexed cellulases.
- the solid circles represent reducing ends, and the open circles represent nonreducing ends.
- Amorphous and crystalline regions are indicated.
- Cellulose, enzymes, and hydrolytic products are not shown to scale.
- Figure 2 depicts a schematic representation of the pCEL5 plasmid constructed in the Example.
- the 2 ⁇ sequence is responsible for episomal replication of the plasmid, and the S. cerevisiae orotidine-5 '-phosphate decarboxylase ⁇ URA3) is used as a selectable marker.
- ENOl?, ENOIj, PGK1 P , PGKh represents the S. cerevisiae enolase 1 and phosphoglycerate kinase 1 promoter and terminator DNA sequences; T.
- EGI represents the endoglucanase I gene of Trichoderma reesei, XYNseq the 23-amino acid secretion signal of the xylanase H gene of T. reesei and S. ⁇ buligera BGLI the ⁇ -glucosidase I gene of Saccharomycopsis fibuligera.
- Figure 3 depicts recombinant S. cerevisiae Y294 strains as plate cultures.
- A SC ' ⁇ RA medium with 20 g.L 1 glucose.
- B YPC medium (10 g.L "1 cellobiose) showing growth of BGLl containing Y294 strains.
- C SC 'URA medium (20 g.L 1 glucose) supplemented with 0.1% CMC; after incubation colonies were washed and the medium was stained with Congo red. CMC degrading Y294 strains (containing EGl) show clearing zones.
- D YP-PASC (10 g.L 1 PASC) medium showing growth of the BGLl, EGl co-expressing strain Y294[CEL5].
- E An enhanced topview of the YP-PASC plate in D to illustrate growth by strain Y294[CEL5]. The plates were photographed after 4 days of incubation at 30 0 C.
- Figure 4 depicts a time course of enzymatic activity of recombinant S. cerevisiae strains (as indicated) on YPD medium.
- A ⁇ -Glucosidase activity, indicated as total activity (supernatant and cell associated — solid symbols) and extracellular activity (supernatant) - open symbols was measured on p-NPG.
- B Extracellular endoglucanase activity was measured on CMC. Symbols used were Y294[RefJ (T, V); Y294[SFI] (A, ⁇ ); Y294[EGI] ( ⁇ , O); Y294[CEL5] (•, O).
- Figure 5 depicts (A) growth curve (solid symbols) and (B) ethanol production
- Figure 6 depicts decreased viscosity of anaerobic YP-PASC cultures at the end of the 240 hour growth period. Viscosity measurements were done over 30 shear rates (2- 200 s "1 ) for the culture media after the growth period as well as for fresh YP-PASC (10 g.L "1 PASC) medium. The average viscosities of the spent culture media were expressed as a percentage of the viscosity of fresh medium.
- Figure 7 depicts a growth curve (solid symbols) of aerobic cultures of recombinant
- One aspect of the present invention relates to host cells containing recombinant enzymes which enable the host cells to derive fermentable sugars from complex polysaccharides found in biomass.
- embodiments of the invention enable digestion and utilization of cellulosic material for fermentation by microorganisms.
- metabolically engineered host cells capable of new biochemical processes that enable growth on cellulosic material as well as concomitant ethanol production are described.
- the embodiments of the invention disclosed herein provide an important step in effectively using biomass material to produce ethanol, which can be subsequently used for a variety of energy needs.
- a "recombinant host cell” as defined herein is a cell which has one or more exogenous genes in the nucleus of the cell.
- the exogenous gene(s) can be carried on a plasmid(s) or integrated into the chromosome(s) of the host cell.
- "Endoglucanase” as defined herein is an enzyme capable of cutting at random within the polysaccharide chain of cellulose to yield predominantly oligosaccharides and is characterized by EC 3.2.1.4 activity.
- ⁇ -glucosidase as defined herein is an enzyme capable of hydrolyzing soluble cellodextrins and cellobiose to glucose units and is characterized by the EC 3.2.1.21 activity.
- Amorphous cellulose is defined herein as a cellulosic fraction that is not in compact crystal structure and can absorb water.
- One aspect of the present invention relates to a recombinant host cell comprising a heterologous polynucleotide which encodes an endoglucanase and a heterologous polynucleotide which encodes a ⁇ -glucosidase, wherein the host cell does not express an exoglucanase, and wherein the host cell can grow on amorphous cellulose as the sole carbon source.
- Another aspect of the present invention relates to a recombinant host cell comprising a heterologous polynucleotide which encodes an endoglucanase and a heterologous polynucleotide which encodes a ⁇ -glucosidase, wherein the host cell can grow on amorphous cellulose as the sole carbon source, and wherein the host cell does not require pre-growth on a non-amorphous cellulose carbon source.
- the recombinant host cell is a yeast.
- the recombinant host cell is of the genus Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces or Yarrowia.
- host cells are yeast of the species S. cerevisiae, S. bulderi,
- the recombinant yeast strains of some aspects of the invention can additionally express other enzymes important for production of ethanol from biomass-derived substrates.
- additional enzymes can be stress-resistance enzymes, such as heat shock proteins, catalases, superoxide dismutases, glutathione reductases and the like.
- enzymes expressed or over-expressed by the host cells in certain aspects of the invention can be enzymes that increase metabolic flux through various metabolic pathways (including the pentose phosphate pathway), alter glucose repression characteristics of the cell, break down or export fermentation inhibitors from the cell, alter the NAD + /NADP + ratio, alter the NAD + /NADH ratio, disrupt the respiratory capacity of the cell, increase growth rate, increase fermentation rate, and/or increase ethanol yield.
- the recombinant yeast strain of certain aspects of the invention can also contain a deletion or disruption of a native enzyme or enzymes in order to increase ethanol yield directly or indirectly.
- enzymes it can be desirable to disrupt or delete in cells in certain aspects of the invention include enzymes which repress the stress response of the cell, alter the flux of carbons away from ethanol production (and thus produce byproducts), allow mating type switching, slow growth, or increase respiratory capacity. Additionally, deletion of enzymatic activities can be useful for creating additional auxotrophic markers which would facilitate the transformation of host cells of certain aspects of the invention.
- cells of certain embodiments can express heterologous enzymes which allow fermentation of pentose sugars or other carbon containing substrates which can be found in the prefermentation biomass of certain aspects of the invention.
- cells of the present invention can be selected through repeated rounds of adaptation experiments to yield more hearty strains suitable for industrial fermentation, and/or cells can be repeatedly crossed to (by mating) cells preselected for good utility in industrial application.
- Progeny cells can then be selected, according to some aspects of the present invention for ability to ferment amorphous cellulose, and further subjected to mating and/or selection to yield increasingly industrially useful progeny. Methods for increasing the "toughness" of a strain and otherwise maximizing its utility for industrial application will be readily apparent to one of skill in the art.
- the endoglucanase and ⁇ - glucosidase can be any suitable endoglucanase derived from, for example, a fungal or bacterial source.
- the endoglucanase can be derived from Holomastigotoides mirabile (e.g. HmEGl, HmEG2 or HmEG3), Humicola grisea (e.g. EGl), Hypocrea pseudokoningii (e.g. EGl), Aspergillus aculeatus (e.g. EGl), Aspergillus kawachii (e.g. cel5A or cel5B), Aspergillus niger (e.g.
- Phanerochaete chrysosporium e.g. Cel5A
- Trichoderma reesei e.g. EGl
- the ⁇ -glucosidase can be derived from, for example, Humicola grisea, Hypocrea pseudokoningii, Aspergillus aculeatus (e.g. bgll), Aspergillus kawachi (e.g. bglA), Aspergillus nidulans, Aspergillus niger (e.g. bgll), Hypocrea jecorina (e.g.
- the endoglucanase(s) can be an endoglucanase I or an endoglucanase II isoform, paralogue or orthologue.
- the endoglucanase expressed by the host cells of the present invention can be recombinant endo-l,4- ⁇ -glucanase.
- the endoglucanase is an endo-1,4- ⁇ -glucanase from Trichoderma reesei.
- the ⁇ -glucosidase is derived from
- the ⁇ -glucosidase is a ⁇ -glucosidase I or a ⁇ -glucosidase ⁇ isoform, paralogue or orthologue.
- the enzymes expressed by the cells of the present invention can be recombinant endo-l,4- ⁇ -glucanase from Trichoderma reesei (teleomorph Hypocreajadinii) and ⁇ -glucosidase from Saccharomycopsis fibuligera source.
- the enzymes can be recombinant endo-l,4- ⁇ -glucanase I
- the recombinant endo-l,4- ⁇ -glucanase I (EGI) from T. reesei and ⁇ -glucosidase I (BGLI) from Saccharomycopsis fibuligera can be recombinantly introduced into an industrial strain of Saccharomyces cerevisiae or another yeast suitable for commercial production of ethanol.
- the recombinant endo-l,4- ⁇ -glucanase I (EGI) from T. reesei and ⁇ -glucosidase I (BGLI) from Saccharomycopsis fibuligera are recombinantly produced by Saccharomyces cerevisiae Y294[CEL5].
- the recombinant enzymes of the present invention can be encoded on a common plasmid or by separate plasmids.
- the plasmids can be high copy plasmids or the more stable lower copy number CEN plasmids.
- the recombinant enzymes of the present invention can be under the control of any of a variety of promoters suitable for their expression including constitutively active promotors or promotors which can be regulated by conditional variables.
- the recombinant enzymes of the present invention can be integrated in to the genomic DNA of the host cell under the control of any suitable promoter.
- the recombinant enzymes can be secreted into the extracellular environment, compartmentalized to various organelles, or tethered to the cell membrane or cell wall.
- the recombinant enzymes can be expressed as fusion proteins.
- the enzymes can be fused to each other directly, or separated by a flexible linker region of amino acids.
- the enzymes of some embodiments of the invention can also be fused to other proteins to promote their secretion, secure them to the cell wall or cell membrane, alter their stability, alter their enzyme kinetics, and/or alter their substrate specificity.
- the enzymes of the present invention can have alternative secretion sequences to affect their secretion kinetics.
- An industrial strain of yeast according to the present invention would also be capable of fermentation of native cellulosics if an exogenous cellulase, notably cellobiohydrolase and/or exoglucanase enzymes, is added to the fermentation broth.
- a recombinant host cell according to the present invention would be useful in combination with other microbes to ferment various substrates found in biomass to ethanol.
- This embodiment of the invention enables fermenting native cellulosics that are present in all plant biomass to ethanol.
- Native cellulosics include both amorphous and crystalline cellulose moieties.
- one aspect of the invention can include the step of hydrolyzing the amorphous and crystalline cellulosics with the addition of cellulase enzymes.
- Another aspect of the invention relates to methods of fermenting amorphous cellulosics which includes the step of hydrolyzing cellulosics by recombinant enzymes produced by the recombiant host cells described herein.
- a method of fermenting amorphous cellulosics to ethanol which includes the steps of hydrolyzing the cellulose chains with endoglucanase and ⁇ -glucosidase produced by a recombinant yeast strain and subsequently recovering the ethanol.
- the method of fermenting amorphous cellulosics includes the steps of hydrolyzing the cellulose chains with endoglucanase and ⁇ -glucosidase co- produced by a single recombinant yeast strain.
- the enzymes are produced by different strains. 40
- the endoglucanase and ⁇ -glucosidase are secreted and can act on amorphose cellulose to yield simple sugars in the reaction medium. The presence of these sugars can be detected by standard assays.
- the cellulosics described herein can be pretreated in a variety of ways known to those skilled in the art to generate amorphous cellulosics susceptible to enzyme hydrolysis by recombinant endoglucanase and ⁇ - glucosidase produced by host cells of the present invention.
- cellulosics in compositions of some aspects of the present invention can be pretreated by steam explosion, ammonium explosion treatment, CO 2 explosion, acid or alkali pretreatment, oxidative pretreatment, organosolvent treatment or enzymatic (cellobiohydrolase) treatment to yield amorphous cellulosics.
- compositions comprising amorphous cellulose together with a host cell capable of converting amorphous cellulose into ethanol via fermentation are also provided.
- Such compositions of the invention can contain amorphous cellulose as the sole carbon source, but alternatively the composition can be supplemented with alternative carbon sources.
- composition of amorphous cellulose and host cells can contain crystalline cellulose as well as additional nutrients suitable for industrial fermentation including, for example, antibiotics, vitamins, minerals, amino acids, salts, pH modifiers, and other ingredients which will be readily apparent to one of skill in the art.
- Phosphoric acid swollen cellulose was prepared as described by Zhang, Y.H. and Lynd, L.R., Biomacromolecules. 6, 1510-1515 (2005) using Avicel PH-IOl (Fluka).
- E. coli strain XLl Blue MRF (Stratagene) was used for plasmid transformation and propagation. Cells were grown in LB medium (5 g.L 1 yeast extract, 10 g.L 1 NaCl, 10 g.L "1 tryptone) supplemented with ampicillin (lOO mg.L "1 ). S.
- fibuligera BGLl were at times cultured on YPC medium (10 g.L " 1 yeast extract, 20 g.L '1 peptone, 20 g.L “1 cellobiose).
- Strain Y294[CEL5] co-expressing the S. fibuligera BGLl and the T. reesei EGl were cultured on YP-PASC medium (10 g/L "1 yeast extract, 20 g.L “1 peptone, 10 g.L “1 PASC).
- Yeast strains were routinely cultured in 250 mL Erlenmeyer flasks containing 100 mL medium at 30 0 C, on a rotary shaker at 100 rpm. For aerobic growth, cultures were grown in 50 mL YP medium containing lOg.L "1 PASC in baffled 250 mL Erlenmeyer flasks on a rotary shaker at 100 rpm and 30 0 C. Cultures were inoculated to ⁇ 2xlO 5 cells per mL from overnight cultures.
- yeast strains were grown in rubber plugged 100-mL glass serum bottles containing 100 mL YP-PASC medium supplemented with 0.01 g.L " ' ergosterol and 0.42 g.L "1 Tween 80 (Yu, S., et al, Appl. Microbiol. Biotechnol. 44, 314-320 (1995)). Precultures of the strains were grown on YPD medium. A precultures of strain Y294[CEL5] was also grown in 10 g.L "1 PASC. For growth on liquid YP-PASC medium three cultures of each strain were inoculated simultaneously. Samples were periodically taken and yeast cells in the media were counted in triplicate on a haemocytometer to produce population growth measurements.
- Y294[REF] S. cerevisiae Y294 (furl: LEU2 ySFI) was designated Y294[SFI] S. cerevisiae Y294 (furl: LEU2 pAZ40) was designated Y294[EG1] S. cerevisiae Y294 (furl: LEm yCEL5) was designated Y294[CEL5]
- S. fibuligera BGLl SEQ ID NO:3
- yeast vector ySFI was previously described (Van Rooyen, R., et al, J. Biotech. 120, 284-295 (2005)).
- yENOl was constructed by replacing the ADH2pj (Alcohol dehydrogenase II promoter and terminator) cassette of pDLGl (La Grange et al. Appl. Environ. Microbiol.
- Sequence verification was done after sequence determination by the dideoxy chain termination method, using an ABI PRISMTM 3100 Genetic Analyzer. Sequence analysis utilized mainly the PC based DNAMAN (version 4.1) package from Lynnon BioSoft and the BLAST program at the National Center for Biotechnology Information (www.ncbi.nih.gov/BLAST/).
- DNA transformation of S. cerevisiae Y294 was performed with the lithium acetate dimethylsulfoxide (DMSO) method described by Hill, J., et al, Nucleic Acid Res. 19, 5791 (1991).
- Autoselective strains were constructed by subsequent transformation with pDFl (La Grange, D.C., et al, Appl. Environ. Microbiol. 62, 1036-1044 (1996)), to ensure maintenance of the £/&43-bearing expression vector under non-selective conditions (Kern, L., et al, Gene 88, 149-157 (1990)); La Grange, D.C., et al, Appl. Environ. Microbiol. 62, 1036-1044 (1996)).
- Saccharomycopsis ⁇ buligera ⁇ -glucosidase BGLl (SEQ ID NO:3) alone (Y294[SFI]), the gene encoding the Trichoderma reesei endoglucanase 1 (EGJ) (SEQ ID NO:1) alone (Y294[EG1]) and a combination of the two genes (Y294[CEL5] - Figure 2).
- BGLl Saccharomycopsis ⁇ buligera ⁇ -glucosidase
- EGJ Trichoderma reesei endoglucanase 1
- Y294[CEL5] a combination of the two genes
- Heterologous expression of the BGLl gene enabled the recombinant strains Y294[SFFJ and Y294[CEL5] to grow on cellobiose as sole carbohydrate source ( Figure 3B).
- Viscosity measurements were done on a Physica MCR 501 (Anton Paar,
- Interval 1 was a pre-shear on the samples at 2 points (30s each, shear rate: 1 s "1 ), interval 2 represented a waiting time before the measurements (5 points, 5s each) and interval 3 was the analysis phase done over 30 points (5s each, shear rate 2-200 s "1 ).
- Table 3 Summary of the reduction if medium viscosity due to PASC degradation, PASC remaining, sugars utilized and ethanol produced by different recombinant Y294 strains under anaerobic fermentations.
- Yeast transformants containing the T. reesei EGl were screened for carboxymethyl-cellulose (CMC) degrading ability by patching on SC 'URA medium containing 0.1% (w/v) CMC (Sigma) with 20 g.L "1 glucose as carbon source. After 48h of growth, colonies were washed of the plate and the remaining CMC was stained with 0.1% Congo red and de-stained with 1% (w/v) NaCl. Extracellular endoglucanase activity was indicated by clearing zone formation. Endoglucanase activity was quantified as described by Bailey, MJ., et al, J. Biotechnol.
- Aerobic cultivations yielded cell counts similar to those found for anaerobic cultivations but in a considerably shorter timeframe (Figure 7). It was previously shown that in aerobic cultivations, S. cerevisiae biomass accumulates to approximately 5 fold the amount that it does in anaerobic cultivations on the same amount of sugar (Van Dijken, J.P., et al, Enzyme Microb. Technol. 26, 706-714 (2000)). 47-
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CA2699460A CA2699460A1 (en) | 2007-09-14 | 2007-09-14 | Method for fermenting cellulosics |
US12/677,956 US20110129888A1 (en) | 2007-09-14 | 2007-09-14 | Method for Fermenting Cellulosics |
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CN103103139A (en) * | 2013-01-29 | 2013-05-15 | 广州分子生物技术有限公司 | Genetic recombination saccharomyces cerevisiae capable of degrading amorphous cellulose |
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WO2012060389A1 (en) * | 2010-11-05 | 2012-05-10 | 旭硝子株式会社 | Transformant of yeast of genus schizosaccharomyces and method for producing same |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN103103139A (en) * | 2013-01-29 | 2013-05-15 | 广州分子生物技术有限公司 | Genetic recombination saccharomyces cerevisiae capable of degrading amorphous cellulose |
CN103103139B (en) * | 2013-01-29 | 2015-06-17 | 广州分子生物技术有限公司 | Genetic recombination saccharomyces cerevisiae capable of degrading amorphous cellulose |
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